Prenylation Inhibitors Block IL-6 and IGF-1 Dependent Activation of RAS Signaling in Multiple Myeloma Cells.

Multiple myeloma (MM) is a fatal hematologic malignancy associated with disruption of RAS-to MAP kinase (ERK) signaling. The IL-6 cytokine family and growth factors such as IGF-1 have been demonstrated to promote malignant plasma cell proliferation through stimulation of ERK and PI-3 kinase/AKT signaling. Prenylation inhibitors such as farnesyltransferase inhibitors (FTIs), geranylgeranyl transferase inhibitors (GGTIs) and the competitive HMG-CoA reductase inhibitor lovastatin have been shown to block RAS post-translational modification and disrupt RAS signaling. To assess efficacy of prenylation inhibitors (e.g. FTI L-744,832, GGTI-2147 and lovastatin) to attenuate MM cell responses to IL-6 and IGF-1, inhibitor-treated cells were analyzed by proliferation/viability assays (MTS) and Western blotting to determine phosphorylated MEK-1/2 and ERK-1/2. FTI L-744,832 was found to inhibit growth of MM cells cultured with IL-6 or IGF-1 even more potently than in cytokine/growth factor-free medium (IC₅₀s 1.3 μM, 1.8 μM and 4.2 μM, respectively). IL-6 moderately protected MM cells from inhibitory effects of GGTI-2147, while IGF-1 had no effect (IC₅₀s 1.1 μM and 0.5 μM vs. 0.5 μM, respectively). IL-6 and IGF-1 protected MM cells from lovastatin-induced growth inhibition (IC₅₀s 4.7 μM and 5.0 μM vs. 1.4 μM, respectively). Furthermore, co-treating MM cells with FTI L-744,832 and GGTI-2147 or lovastatin synergistically inhibited proliferation of MM cells regardless of the presence or absence of IL-6 or IGF-1. Western blotting demonstrated that FTI/GGTI or FTI/lovastatin co-treatment more completely blocked activation of MEK-1/2 and ERK-1/2 in NCI-H929 cells than treatment with any of the compounds alone. Co-treatment also elicited greater inhibition of IL-6 and IGF-1 induced MEK-1/2 and ERK-1/2 activation in NCI-H929 cells. IL-6, IGF-1 and prenylation inhibitors had no effects on AKT phosphorylation status in NCI-H929 cells. To evaluate the clinical relevance of these observations, primary MM cells were obtained from bone marrow aspirates (n=6) or from peripheral blood of a patient with plasma cell leukemia/MM (PCL/MM). Primary MM cells were isolated by magnetic cell sorting using CD138-coupled microbeads, titrated with prenylation inhibitors in the presence of IL-6 and synergy was calculated using the CalcSyn program. Activating RAS mutations (4 K-RAS, 1 N-RAS) were found in 4/7 (57%) MM patient samples, with one sample harboring both K- and N-RAS mutations. FTI L-744,832 elicited anti-myeloma effects only at concentrations much higher than those found to inhibit healthy donor CD34+ cells (IC₅₀’s 51–396 μM vs. 8.2μM) or MM cell lines (1.1–23.8μM) and thus may be ineffective or cause non-specific toxicity when used as a single agent. However, IC₅₀’s calculated for GGTI-2147 and lovastatin were generally higher for CD34+ cells as compared to primary MM cells, supporting specificity of these compounds. Furthermore, combination of FTI with GGTI or lovastatin synergistically inhibited primary MM cell proliferation (IC₅₀’s 0.6–23.1 μM). Our results support that inhibition of RAS down-stream signaling is a major mechanism through which FTI/GGTI and FTI/lovastatin co-treatment synergistically inhibit MM cell proliferation, even in the presence of IL-6 and IGF-1. Incomplete response to FTI treatment may be explained by alternative prenylation of K- and N-RAS by GGTase I in the presence of FTIs. As the majority of RAS mutations in MM occur in K- and N-RAS, FTI-resistance due to alternative geranylgeranylation may have therapeutic consequences.